Project Summary: The immune system responds rapidly to traumatic injuries in multiple ways, as cells and mediators of the innate and adaptive immune systems undergo changes that have been categorized as both pro- inflammatory and counter-inflammatory immune responses. We propose that while this immune response may have evolved to protect the injured host from infections or excessive reactivity to damaged tissues, these contrasting responses ultimately disrupt immune system homeostasis. The loss of homeostasis is central to the immunological complications associated with traumatic injuries. In addition, injuries have been shown to cause a two-hit response phenotype, defined as an amplified secondary inflammatory response in individuals that survive traumatic injuries. This ?second hit? usually is an infection and can lead to septic shock and multiple organ failure (MOF). Thus, the combined risk of developing infections and the two-hit response is a principal complication of traumatic injuries. Since trauma creates a complex physiological response, specific treatments targeting the immune system to enhance anti-microbial immunity and promote resolution or healing have not yet been developed for clinical use. However, advances have been made in pre-clinical animal studies to suggest that treatment with immune response modulators (IRMs) can redirect the injury response to enhance immune function and protect injured animals from developing post-injury infections. The goal of the proposed studies will be to use systems biology approaches to phenotype how a specific class A CpG oligodeoxynucleotide (ODN), CpG2336, or IL-12p75 treatment changes the immune response to injury. Cellular time of flight mass cytometry (CyTOF) will be used to specifically measure immune cell changes in blood, spleen, and lymph nodes of sham or burn mice treated with these IRMs. Our approach for CyTOF experiments will be to treat sham or burn mice with CpG2336 or IL-12. Fluorescent tagged IRMs will be given to mice that will be harvested at 30, 90, and 120 minutes after administration to identify early cellular targets of IRMs, and these cells will be isolated in subsequent experiments and profiled with CyTOF and Luminex assays to evaluate activation markers and cytokine production. At day 7 after injury, blood, lymph node, and spleen cells will be prepared for CyTOF staining using panels to broadly identify immune cell subsets and cell activation phenotypes. We will then use CyTOF to evaluate IRM treatment effects on cellular responses to S. pneumoniae infection in normal and injured mice. These experiments together will help phenotype the immune response to injury and demonstrate the effects of IRM treatment on survival in a post-trauma infection model.